Cryopreservation (that is, preservation at very low temperatures) of organs would allow organ banks to be established for transplant surgeons in much the same way blood banks serve the medical community today. The main difficulty with cryopreservation is that it requires the perfusion of organs with high concentrations of cryoprotective agents (water soluble organic molecules that minimize or prevent freezing injury during cooling to very low temperatures). No fully suitable equipment or process has been developed to date for carrying out this perfusion process. This has prevented the establishment of viable organ banks that could potentially save lives.
Devices and methods for perfusing organs with cryoprotectant have been described in the literature since the early 1970's. See, Pegg, D. E., Banking of Cells, Tissues, and Organs at Low Temperatures, Current Trends in Cryobiology, A. U. Smith, Editor, Plenum Press, New York, 1970: pp. 153-180, but particularly pages 175-177; and Pegg, D. E., Perfusion of Rabbit Kidneys with Cryoprotective Agents, Cryobiology 9:411-419, 1972).
In the apparatus initially described by Pegg, two perfusion circuits operated simultaneously, one with and one without cryoprotectant. Cryoprotectant was introduced and removed by abruptly switching from the cryoprotectant-free circuit to the cryoprotectant-containing circuit, then back again. The pressure was controlled by undescribed techniques, and data was fed into a data logger which provided a paper tape output which was processed by a programmable desk-top Wang calculator. The experimental results were poor. The equipment and technique described were considered inadequate by Pegg and his colleagues, who later modified them considerably.
In 1973, G. J. Sherwood and J. R. Flower, in: Organ Preservation (D. E. Pegg, editor, Churchill Livingstone, London, 1973, pp. 152-174), described four potential perfusion systems, none of which are known to have been built, The first system consisted of a family of reservoirs connected directly to the organ via a multiway valve, changes being made in steps simply by switching from one reservoir to another.
The second system created changes in concentration by metering flow from a diluent reservoir and from a cryoprotectant concentrate reservoir into a mixing chamber and then to the kidney. No separate pump for controlling flow to the kidney was included. Total flow was controlled by the output of the metering pumps used for mixing. A heat exchanger was used before rather than after the filter, and there was an absence of any arterial sensing. As will become readily apparent below, the only similarity between this system and the present invention was the use of two concentration sensors, one in the arterial line and one in the venous line of the kidney. Organ flow rate was forced to vary in order to minimize A-V concentration differences. The sensing of concentration before and after the kidney in the circuit is analogous to but substantially inferior to the use of a refractometer and a differential refractometer in the present invention. The present inventors' experience has shown that the use of a differential refractometer is necessary for its greater sensitivity. The concept of controlling organ A-V gradient by controlling organ flow is distinctly inferior to the system of the present invention.
The third system described by Sherwood et al. also lacked a kidney perfusion pump, relying on a "backpressure control valve" to recirculate perfusate from the filter in such a way as to maintain the desired perfusion pressure to the kidney. As with the second Sherwood system, the heat exchanger is proximal to the filter and no bubble trap is present. The perfusate reservoir's concentration is controlled by metered addition of cryoprotectant or diluent as in the second Sherwood system, and if flow from the organ is not recirculated, major problems arise in maintaining and controlling perfusate volume and concentration. None of these features is desirable.
The fourth system was noted by Pegg in an appendix to the main paper. In this system, perfusate is drained by gravity directly from the mixing reservoir to the kidney through a heat exchanger, re-entering the reservoir after passing through the kidney. Concentration is sensed also by directly and separately pumping liquid from the reservoir to the refractometer and back.
Modifications and additional details were reported in 1977 (Pegg, D. E., and Wusteman, M. T., Perfusion of Rabbit Kidneys with Glycerol Solutions at 5.degree. C.). The apparatus used one mixing reservoir and one reservoir for adding glycerol concentrate or glycerol-free perfusate to the mixing reservoir to control concentration. The volume of the mixing reservoir was held constant during perfusion, necessitating an exponentially increasing rate of diluent addition during cryoprotectant washout to maintain a linear rate of concentration change. The constant mixing reservoir volume and the presence of only a single delivery reservoir also made it impossible to abruptly change perfusate concentration. All components of the circuit other than the kidney and a pre-kidney heat exchanger were located on a lab bench at ambient temperature, with the reservoir being thermostatted at a constant 30.degree. C. The kidney and the heat exchanger were located in a styrofoam box whose internal temperature was not controlled. Despite this lack of control of the air temperature surrounding the kidney, only the arterial temperature but not the venous temperature or even the kidney surface temperature was measured. The use of a styrofoam box also did not allow for perfusion under sterile conditions. The only possible way of measuring organ flow rate was by switching off the effluent recirculation pump and manually recording the time required for a given volume of fluid to accumulate in the effluent reservoir, since there was no perfusion pump which specifically supplied the organ, unlike the present invention. Pressure was controlled, not on the basis of kidney resistance, but on the basis of the combined resistance of the kidney and a manually adjustable bypass valve used to allow rapid circulation of perfusate through the heat exchanger and back to the mixing reservoir. The pressure sensor was located at the arterial cannula, creating a fluid dead space requiring manual cleaning and potentially introducing undesired addition of unmixed dead space fluid into the arterial cannula. Pressure control was achieved by means of a specially-fabricated pressure control unit whose electrical circuit was described in an earlier paper (D. E. Pegg and C. J. Green, Renal Preservation by Hypothermic Perfusion. 1. The importance of pressure-control, Cryobiology 10:56-66, 1973). Arterial concentration but not venous concentration was measured. No computer control or monitoring was used. Concentration was controlled by feeding the output of the recording refractometer into a "process controller" for comparison to the output of a linear voltage ramp generator and appropriate adjustment of concentrate or diluent flow rate. Glycerol concentrations were measured manually at 5 minute intervals at both the mixing reservoir and the arterial sample port: evidently, the refractometer was not used to send a measurable signal to a recording device. Temperature and flow were recorded manually at 5 minute intervals. Arterial pressure and kidney weight were recorded as pen traces on a strip chart recorder. None of these features is desirable.
Further refinements were reported by Jacobsen, I. A., Pegg, D. E., Wusteman, M. C., and Robinson, S. M., Transplantation of Rabbit Kidneys Perfused with Glycerol Solutions at 10.degree. C., Cryobiology 15:18-26, 1978. A bubble trap was added, the sample port on the kidney bypass was eliminated (concentration was measured at the distal end of the bypass line instead), and temperature was recorded as a trace on a strip chart recorder rather than manually every 5 minutes. Additionally, these authors reported that bypass concentration lagged reservoir concentration by 5 min (v. 3 min or less for arterial concentration in the present invention) and that terminal cryoprotectant concentration could not be brought to less than 70 mM after adding 5 liters of diluent to the mixing reservoir (v. near-zero terminal concentrations in the present invention using less than 3 liters of diluent and using peak cryoprotectant concentrations approximately twice those of Jacobsen et al.).
A variation on the system was also reported the same year by Jacobsen (Jacobsen, I. A., Distribution and Removal of Glycerol by Vascular Albumin Perfusion in Rabbit Kidneys, Cryobiology 15:302-311, 1978). Jacobsen measured but did not report air temperatures surrounding the kidney during perfusion. He reduced the mixing reservoir volume to 70 ml, which was a small fraction of the 400 ml total volume of the circuit. No electronic-output refractometer appears to have been used to directly sense glycerol concentration and control addition and washout. Instead, the calculated values of concentrate or diluent flow rate were drawn on paper with India ink and read by a Leeds and Northrup Trendtrak Programmer which then controlled the concentrate/diluent pump. Despite the low circuit volume, the minimum concentration of cryoprotectant which could be achieved was about 100 mM.
Additional alterations of the same system were reported by Armitage et al. in 1981 (W. J. Armitage, G. Matthes, and D. E. Pegg, Seleno-dl-methionine Reduces Freezing Injury in Hearts Protected with Ethanediol, Cryobiology 18:370-377, 1981). Essentially, the entire perfusion circuit previously used was placed into a refrigerated cabinet. Instead of a voltage ramp controller, a cam-follower was used. Again, however, it was necessary to calculate the required-rates of addition of glycerol or diluent using theoretical equations in order to cut the cam properly, an approach which may introduce errors in the actual achievement of the desired concentration-time histories. Finally, a modification was made in which an additional reservoir was added to the circuit. This reservoir was apparently accessed by manual stopcocks (the mode of switching to and from this reservoir was not clearly explained), and use of the new reservoir was at the expense of being able to filter the perfusate or send it through a bubble trap. The new reservoir was not used to Change cryoprotectant concentration; rather, it was used to change the ionic composition of the medium after the cryoprotectant had been added. The volume of the mixing reservoir was set at 500 ml, allowing a final cryoprotectant concentration of 40 mM to be achieved.
The circuits described above represent the current state of the art of cryoprotectant perfusion by others known to the present inventors.
An approach to organ preservation at cryogenic temperatures previously described by the present inventors involved vitrifying rather than freezing organs during cooling. Vitrification, or solidification without freezing, can be brought about in living systems by replacing large fractions of water in these systems with cryoprotectant agents (also known as cryoprotectants) whose presence inhibits crystallization. In known techniques, however, it has never been possible to use sufficiently high cryoprotectant concentrations without killing the organ. Vitrification typically requires concentrations greater than 6 molar cryoprotectant, whereas the limiting concentration for organ survival is typically about 4 molar.
One type of damage potentially caused by cryoprotectants is osmotic damage. Cryobiologists learned of the osmotic effects of cryoprotectants in the 1950's and of the necessity of controlling these effects so as to prevent unnecessary damage during the addition and removal of cryoprotectants to isolated cells and tissues. Similar lessons were leaned when cryobiologists moved on to studies of whole organ perfusion with cryoprotectants. Attention to the principles of osmosis were essential to inducing tolerance to cryoprotectant addition to organs. Yet despite efforts to control deleterious osmotic effects of cryoprotectants, limits of tolerance to cryoprotectants are still observed. There appear to be genuine, inherent toxic effects of cryoprotectants that are independent of the transient osmotic effects of these chemical agents.
Studies by the present inventors and others have examined methods of controlling the non-osmotic, inherent toxicity of cryoprotective agents. The results indicate that several techniques can be effective alone and in combination. These include (a) exposure to the highest concentrations at reduced temperatures; (b) the use of specific combinations of cryoprotectants whose effects cancel each other's toxicities; (c) exposure to cryoprotectants in carrier solutions that are optimized for those particular cryoprotectants; (d) the use of non-penetrating agents that can substitute for a portion of the penetrating agent otherwise needed, thus sparing the cellular interior from exposure to additional intracellular agent; and (e) minimization of the time spent within the concentration range of rapid time-dependent toxicity. Means by which these principles could be applied to whole organs so as to permit them to be treated with vitrifiable solutions without perishing, however, have not been clear or available.
Some of these techniques are in potential conflict with the need to control osmotic forces. For example, reduced temperatures also reduce the influx and efflux rate of cryoprotectants, thereby prolonging and intensifying their osmotic effects. Similarly, minimizing exposure time to cryoprotectants maximizes their potential osmotic effects. Thus, there must be a balance reached between the control of osmotic damage and the control of toxicity. Adequate means for obtaining this balance have not been described in the literature. It is also true that, in some cases, intensifying osmotic effects of cryoprotectants by minimizing exposure times to these agents can be beneficial and complementary to the reduced toxicity that results, but safe means for achieving this in whole organs have not been described.
Organ preservation at cryogenic temperatures would permit wastage of valuable human organs to be considerably reduced and would facilitate better matching of donor and recipient, a factor which continues to be important despite the many recent advances in controlling rejection. See, Takiff, H., et al., Transplantation 47:102-105 (1989); Gilks, W. R., et al., Transplantation 43:669-674 (1987). A recent approach to the induction of tolerance to transplanted organs requires 10-200 days for the host immune system to be "re-educated" to accept the graft as "self", a time that can only be attained by being able to cryopreserve the cadaver organ See, Posselt, A. M., et al., Science 249:1293-1295 (1990); Remuzzi, G., et al., The Lancet 337:750-752 (1991).
One major limitation in organ cryopreservation studies has been the lack of suitable equipment for controlling perfusion parameters such as cryoprotectant concentration-time history, pressure, and temperature. Previously described standard perfusion machines are not designed for this application and are unable to meet the requirements addressed here. Patented techniques heretofore known are described in:
U.S. Pat. No. 3,753,865 to Belzer et al.
U.S. Pat. No. 3,772,153 to De Roissart et al.
U.S. Pat. No. 3,843,455 to Bier
U.S. Pat. No. 3,892,628 to Thorne, G. H., et al.
U.S. Pat. No. 3,914,954 to Doerig, R. K.
U.S. Pat. No. 3,995,444 to Clark et al.
U.S. Pat. No. 4,629,686 to Gruenberg, M. L.
U.S. Pat. No. 4,837,390 to Reneau, R. P.
Equipment described for cryopreservation applications in the past have permitted only relatively simple experimental protocols to be carried out, and have often been awkward to use. Only Adem and Harness have reported using a computer for organ perfusion with cryoprotectant See, Adem, C. G., et al., J. Biomed. Engineering 3:134-139, 1981. However, their specific design has several major flaws that limits its utility.
The present invention overcomes substantially all of the deficiencies of known apparatus and methods.